CN114231645B - SNP molecular marker related to black peritoneum characters of chickens and application - Google Patents
SNP molecular marker related to black peritoneum characters of chickens and application Download PDFInfo
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Abstract
The invention discloses a SNP molecular marker related to black peritoneum characters of chickens and application thereof. Relates to the technical field of chicken marking auxiliary selection. The invention classifies the black peritoneum character, detects the NDV antibody response and carries out genotyping. The hatching batch, sex, and first three principal components were used as fixation effects for GWAS analysis. Obtaining a remarkable SNP locus (rs 317439499), and then carrying out association analysis to obtain that the allele A has a remarkable negative effect on black peritoneal characters and a remarkable positive effect on the response level of newcastle disease antibodies; it is obtained that allele G has a very significant positive effect on the black peritoneal trait and a significant negative effect on the newcastle disease antibody response level. The molecular marker SNP locus can be used for molecular marker assisted breeding of black peritoneum characters of chickens. The genetic marker can reduce the number of black peritoneal individuals in the population and improve the response level of newcastle disease antibodies.
Description
Technical Field
The invention relates to the technical field of chicken marker assisted selection, in particular to a SNP molecular marker related to black peritoneum characters of chickens and application thereof.
Background
Chicken is the second most consumed meat in China, and in recent years, the average consumption of chicken in China is continuously growing. To prevent epidemic disease transmission and ensure the quality safety of live poultry products, the high-quality broiler industry mainly dealing with live poultry is gradually changed into the iced fresh and slaughter processing type. Therefore, the breeding of the Tu Tiwai-viewing characters of the broiler chickens, such as black peritoneal characters, which directly affect the economic benefits of the breed, is emphasized.
The black peritoneal trait of chickens appears to be a dark blue appearance of abdominal skin, with black connective tissue appearing when the skin is removed. It severely affects carcass appearance and affects economic efficiency. And the black abdominal character is common in yellow feather broilers, and 5% -95% of Huiyang beard chickens have black peritoneum at 3 weeks of age, but the research on the black peritoneum character of chickens is less at present.
Therefore, how to provide a SNP molecular marker related to black peritoneal traits of chickens is a problem to be solved by the person skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a SNP molecular marker related to black peritoneum characters of chickens and application thereof. The black peritoneum trait of the chicken is taken as a moderate genetic trait, a certain improvement space exists, the black peritoneum trait of the chicken is taken as a complex trait, the black peritoneum trait of the chicken is suitable for searching SNP (single nucleotide polymorphism) obviously associated with the trait by using GWAS (whole genome association analysis), and the found obvious SNP locus is expected to be applied to subsequent breeding of the black peritoneum trait of the chicken.
In addition, there was a moderate negative genetic correlation of the black peritoneum with newcastle disease antibody responses-0.42. Genetic association generally results from polygenic effects, however genetic association may also result from linkage disequilibrium between different loci. The present invention employs a multiple trait GWAS to identify multiple gene loci, and the resulting marker trait associations are referred to as cross-phenotype (CP) associations. Generally, the higher the genetic correlation between the traits studied, the higher the efficacy of the multiple trait GWAS. Through the multi-trait GWAS we can screen SNPs with antagonism, i.e. SNPs have a positive effect on one trait and another trait has a negative effect. Such SNP sites are used for molecular marker assisted breeding of chickens, and we can obtain chickens with both low black peritoneum and high newcastle disease antibody response level. .
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a molecular marker related to black peritoneum character of chicken is characterized in that SNP locus of molecular marker is positioned at 101 th position shown in nucleotide sequence SEQ ID NO.1, and is A or G.
The invention also provides a screening method of the molecular marker related to the black peritoneum character of the chicken, which comprises the following steps:
(1) Determining black peritoneum and newcastle disease antibody response level data of the test chicken;
(2) Extracting DNA of a test chicken;
(3) Carrying out GWAS analysis through an mvLMM model to obtain a remarkable SNP locus;
(4) Verifying the significant SNP locus by haplotype and phenotype association analysis;
(5) Significant SNP sites were obtained.
The invention also provides application of the molecular marker related to the black peritoneum character of the chicken in chicken strain breeding.
Preferably: when the SNP locus is A, the chicken has lower black peritoneum and higher newcastle disease antibody response level.
Preferably: the method comprises the following steps:
(1) Extracting genome DNA;
(2) Detecting and testing the genotype of the SNP locus of the molecular marker;
(3) And selecting the test chicken with genotype A/A for breeding dominant strains.
The invention also provides application of the molecular marker in preparation of a detection kit.
The beneficial effects are that: the dominant strain is mainly characterized by low black peritoneal extent and high newcastle disease antibody response level.
Compared with the prior art, the invention discloses SNP molecular markers related to the black peritoneum character of chickens and application thereof, and the obtained technical effects are that the black peritoneum character is classified, serum is separated, NDV antibody response is detected by adopting an indirect ELISA method, and the wing venous blood is subjected to genotyping by simplifying a genome technology. Quality control was performed with the criteria that only gene markers covering 70% of individuals were retained, deleting markers with MAF less than 0.01. The hatching lot, sex, first three principal components were used as fixed effects for GWAS analysis using a multivariate linear mixture model mvLMM (multivariate linear mixed models) in GEMMA. By analyzing the GWAS result, a remarkable SNP locus rs317439499 is obtained, and an allele A of the locus has a negative effect on black peritoneal characters and a positive effect on the response level of newcastle disease antibodies. Carrying out correlation analysis on the SNP, the black peritoneal trait and the newcastle disease antibody response level by using R language package haplo.stats, so as to obtain that the allele A has a very obvious negative effect on the black peritoneal trait and a obvious positive effect on the newcastle disease antibody response level; it is obtained that allele G has a very significant positive effect on the black peritoneal trait and a significant negative effect on the newcastle disease antibody response level. The molecular marker SNP locus rs317439499 can be used for molecular marker assisted breeding of black peritoneum characters of chickens. The genetic marker can reduce the number of black peritoneal individuals in the population and improve the response level of newcastle disease antibodies.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a drawing showing a hierarchical standard chart of black peritoneal properties provided by the invention, wherein the hierarchical standard chart is respectively of level 0, level 1 and level 2 from left to right.
FIG. 2 is a schematic representation of Manhattan provided by the present invention, showing that a significant SNP site is found on chromosome one.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses an SNP molecular marker related to black peritoneum characters of chickens and application thereof.
Example 1
1 test animal:
the test population used was F, institute of animal science, academy of agricultural sciences, guangdong province 2 The parent of the hybrid resource group of the yellow-feather broiler is a yellow-feather broiler quick and large male parent A line and a slow Huiyang beard chicken H line, wherein the A line mainly uses the growth character as a main target to select and select over 10 generation blocking groups for secondary breeding, and the H line is a Guangdong local variety and is native to the Guangdong Huiyang region. The yellow feather broiler resource group F 2 The generation is divided into 6 batches of hatching and hatching.
Example used together 395F with phenotype 2 The generation of individuals, 212 hens, 183 hens, were from 8 half-sibling families. Two immunizations were performed with commercial newcastle disease virus LaSota strain live vaccine at 25 and 50 days of age, respectively. Slaughter at 91 days old, according to the prior study, the ratio of melanin deposition area on peritoneum and black as shown in FIG. 1The black peritoneum character is graded by the color of the element deposition area, serum is separated, and the NDV antibody response is detected by an indirect ELISA method, and the specific operation is as follows:
live vaccines of commercial newcastle disease virus LaSota strain (international b.v., boxmeer, netherlands) were immunized at standard doses given in the vaccine specifications at 25 and 50 days of age, respectively. Serum samples were collected from chickens 6 weeks after the second immunization (91 days old). NDV antibody responses were detected by indirect ELISA according to the instructions of NDV antibody detection kit manufactured by BioCheck, foster, california, usa and expressed as S/P values of the corresponding dilutions. The fin venous blood is genotyped by simplifying genomic techniques.
2 test method
2.1 genotyping:
genomic DNA was extracted from venous blood by phenol/chloroform method for genotyping. Autosomal data from 1 to 28 were used for the data in the examples. Simplified genome signature typing 10X deep sequencing was performed using the SLAF method, leaving only the gene signature covering 70% of individuals, and then deleting the signature with MAF less than 0.01. 115706 SNP markers are obtained after quality control.
2.2GWAS：
The GWAS was realized by the mvLMM model of software GEMMA, and the two personalities of black peritoneum and newcastle disease antibody response level were used as study traits. The mvLMM statistical model is as follows:
y＝Wv+Xβ+Zu+e
where y represents the phenotype matrix of n x t, n is the number of individuals, t is the number of traits studied, v marks the effect, and β is the fixed effect, including the first three principal components. u is a polygenic effect, consistent with NDistribution of->And (3) calculating a genetic relationship matrix for the multigenic effect variance and the K-mark effect. e is the random residual, obeying N +.>Distribution W, X, Z corresponds to the design matrix.
A significant level of genome was obtained using a conservative bonferrori correction method, thereby reducing the false positive rate, i.e. p=0.05/N, N being the number of effective SNP sites. The P-value threshold for this genomic significance level was 4.32X10 -7 (0.05/115706). The manhattan diagram is drawn using the R language CMplot package.
2.3 association analysis of SNPs with phenotypes
Selected SNPs were analyzed for association with black peritoneal traits and newcastle disease antibody response levels, respectively, using R-language package haplo. The software package may calculate score statistics to evaluate the association of traits with SNPs.
3 results and analysis
By the method of F 2 395 individuals of the filial resource population of the yellow-feather broilers are subjected to multi-character GWAS analysis, and a significant SNP locus rs317439499 is screened out. At Chromosome1:49767755 (version of the gene, gallus_gap-5.0), the polymorphisms of this molecular marker are A and G.
The specific molecular marker is located at position 101 of the following nucleotide sequence:
GCCTGCAGCCACAGTTAAATGTTTTATCCCCGCCCCTTTGGCTCAGCCAATATGTGGGAGAAAGAGGAAAGAATTGGATATATTGGATACAATCCTGTTCA (G) CTGCTTTAGGTTGTGATGAGCTAACAACAAGTGCCACATTTCTTGCTCATACGATGGCAGCTATGAAAGTAATGCCTCTTACTTTATGATGCTGGCCCAT, as shown in SEQ ID NO. 1.
The manhattan diagram obtained by the GWAS is shown in figure 2, and then the correlation analysis is carried out on the selected SNP and the two characters of the study by using an R language package hard. Stats, the specific analysis results are shown in the table 1 and the table 2, so that the allele A has a very obvious negative effect on the black peritoneal character and a obvious positive effect on the newcastle disease antibody response level; it is obtained that allele G has a very significant positive effect on the black peritoneal trait and a significant negative effect on the newcastle disease antibody response level.
Table 1 black peritoneal correlation analysis
TABLE 2 Newcastle disease antibody response level correlation analysis
In conclusion, the test chicken with genotype A/A at SNP locus rs317439499 can be selected for breeding the dominant strain, and the dominant strain is mainly represented by low black peritoneal extent and high newcastle disease antibody response level.
The invention utilizes the multi-trait GWAS to prove the genetic antagonism between the two traits of chicken black peritoneum and newcastle disease antibody response on the whole genome level.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> institute of animal science at the national academy of agricultural sciences in Guangdong province
<120> SNP molecular marker related to black peritoneum character of chicken and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 201
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (101)..(101)
<223> n=a or G
<400> 1
gcctgcagcc acagttaaat gttttatccc cgcccctttg gctcagccaa tatgtgggag 60
aaagaggaaa gaattggata tattggatac aatcctgttc nctgctttag gttgtgatga 120
gctaacaaca agtgccacat ttcttgctca tacgatggca gctatgaaag taatgcctct 180
tactttatga tgctggccca t 201
Claims (3)
1. The application of the SNP molecular marker detection reagent related to the black peritoneum character of the chicken in chicken strain breeding is characterized in that a test chicken with the genotype of the SNP molecular marker locus being A/A is selected, and the breeding of a dominant strain is carried out, wherein the dominant strain is low in black peritoneum degree and high in newcastle disease antibody response level; the SNP molecular marker locus is positioned at the 101 st position shown in the nucleotide sequence SEQ ID No.1 and is A or G; the test chickens are F2 generation hybrid of yellow feather chickens and Huiyang beard chickens.
2. The use of claim 1, wherein when the SNP molecular marker site is a, the chicken has a lower black peritoneum and a higher newcastle disease antibody response level.
3. The use according to claim 2, comprising the steps of:
(1) Extracting genome DNA;
(2) Detecting the genotype of the SNP molecular marker locus;
(3) And selecting the test chicken with genotype A/A for breeding dominant strains.
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